PROJECT SUMMARY/ABSTRACT Enterococci are a leading cause of hospital-acquired infections in the USA, where they are frequent opportunistic pathogens of the catheterized urinary bladder and wounds. Enterococci are found in up to 70% of wound infections, often in association with multiple genera growing within biofilms. There is a knowledge gap in our understanding of how Enterococcus faecalis contributes to the pathogenesis of polymicrobial infections. The long term goal of this research is to identify, define, and harness mechanisms by which E. faecalis modulates its environment to alter the pathogenesis of polymicrobial infections in vivo. The specific goal of this exploratory R21 research grant is to identify factors that mediate E. faecalis and E. coli interactions in a murine wound infection model. The hypothesis under investigation is that E. faecalis changes the local environmental during polymicrobial infection by exporting molecules that cue E. coli virulence. This hypothesis is based on preliminary data showing that E. faecalis enhances the growth and virulence of E. coli within polymicrobial biofilms in vitro under iron-limited conditions and in vivo during wound infection. To address this hypothesis and achieve the goals of this proposal, screens for both the E. faecalis effector molecules and the E. coli responsive pathways will be performed in the following specific aims: 1) Identify the E. faecalis factors that augment E. coli biofilm growth, and 2) Identify the E. coli genes required for augmented growth in response to E. faecalis cues. Upon completion of these aims, the expected outcomes of this project will include the identification of all of the non-essential genes that are involved in E. faecalis + E. coli synergy during infections. The findings in this study are important because they will be the first to identify E. faecalis factors that promotes chronic, polymicrobial, biofilm-associated infections. The approach takes advantage of an innovative in vitro platform that mimics aspects of the in vivo infection environment to specifically examine pathways of microbial synergy conferred by E. faecalis within the iron-restricted host. Altogether, this work holds great promise for 1) understanding the molecular basis of polymicrobial synergies that occur during infection, and 2) identifying new anti-infective and therapeutic targets to treat increasingly common and often drug-resistance polymicrobial infections.